1. Field of the Invention
The present invention provides a method of increasing salt tolerance in a plant by overexpressing a gene encoding a mutant SOS2 protein in at least one cell type in the plant. The present invention also provides for transgenic plants expressing the mutant SOS2 proteins.
2. Discussion of the Background
Soil salinity is a serious environmental stress limiting plant productivity. Sodium ions (Na+), which are abundant in saline soils, are cytotoxic in plants when they accumulate to high concentrations. Na+ enters plant cells through transporters such as HKT1 (Rus et al., 2001) and nonselective cation channels (Amtmann and Sanders, 1999). To prevent Na+ buildup in the cytoplasm, plant cells employ Na+/H+ antiporters at the plasma membrane and tonoplast to transport Na+ into the apoplast and vacuole, respectively (Apse et al., 1999; Qiu et al., 2002). Overexpression of the Arabidopsis thaliana plasma membrane Na+/H+ antiporter Salt Overly Sensitivel (SOS1) or the vacuolar Na+/H+ antiporter AtNHX1 improves salt tolerance in transgenic plants (Apse et al., 1999; Zhang and Blumwald, 2001; Zhang et al., 2001; Shi et al., 2003). Enhanced salt tolerance can also be achieved by overexpression of the vacuolar H+-pyrophosphatase AVP1, which generates the driving force for Na+ transport into the vacuole (Gaxiola et al., 2001).
Recently, a regulatory pathway for ion homeostasis and salt tolerance was identified in A. thaliana (Zhu, 2000, 2002). Salt stress is known to elicit a rapid increase in the free calcium concentration in the cytoplasm (Knight et al., 1997). SOS3, a myristoylated calcium binding protein, is proposed to sense this calcium signal (Liu and Zhu, 1998; Ishitani et al., 2000). SOS3 physically interacts with the protein kinase SOS2 and activates the substrate phosphorylation activity of SOS2 in a calcium-dependent manner (Halfter et al., 2000; Liu et al., 2000). SOS3 also recruits SOS2 to the plasma membrane, where the SOS3-SOS2 protein kinase complex phosphorylates SOS1 to stimulate its Na+/H+ antiport activity (Qiu et al., 2002; Quintero et al., 2002). Loss-of-function mutations in SOS3, SOS2, or SOS1 cause hypersensitivity to Na+ (Zhu et al., 1998).
SOS2 has a highly conserved N-terminal catalytic domain similar to that of Saccharomyces cerevisiae SNF1 and animal AMPK (Liu et al., 2000). Within the SOS2 protein, the N-terminal catalytic region interacts with the C-terminal regulatory domain (Guo et al., 2001). SOS3 interacts with the FISL motif in the C-terminal region of SOS2 (Guo et al., 2001), which serves as an auto-inhibitory domain. A constitutively active SOS2 kinase, T/DSOS2, can be engineered by a Thr168-to-Asp change (to mimic phosphorylation by an upstream kinase) in the putative activation loop. The kinase activity of T/DSOS2 is independent of SOS3 and calcium (Guo et al., 2001). Removing the FISL motif (SOS2DF) or the entire C-terminal regulatory domain (SOS2/308) may result in constitutively active forms of SOS2 (Guo et al., 2001; Qiu et al., 2002). The activation loop mutation and the autoinhibitory domain deletions have a synergistic effect on the kinase activity of SOS2, and superactive SOS2 kinases T/DSOS2/308 or T/DSOS2/DF can be created when the two changes are combined (Guo et al., 2001; Qiu et al., 2002). The present inventors have shown that T/DSOS2/DF could activate the transport activity of SOS1 in vitro, whereas the wild-type SOS2 protein could not (Qiu et al., 2002). However, at the time of the present invention, whether these active forms of SOS2 can function in vivo was not known.
In the trophic chain, plant roots play pivotal roles by taking up mineral nutrients from soil solutions. Plant roots experience constant fluctuations in soil environments. A frequent variant in the soil solution is Na+ concentration (Epstein et al., 1980). Na+ is not an essential ion for most plants. In fact, the growth of the majority of plants, e.g., glycophytes, is inhibited by the presence of high concentrations of soil Na+. External Na+ causes K+ deficiency by inhibiting K+ uptake into plant cells (Wu et al., 1996). Na+ accumulation within the cell is toxic to many cytosolic enzymes. In contrast, many cellular enzymes are activated by K+, which is the most abundant cation in the cytoplasm. Certain cytoplasmic enzymes are especially prone to Na+ inhibition when K+ concentration is reduced (Murguia et al., 1995). Therefore, maintaining intracellular K+ and homeostasis to preserve a high K+/Na+ ratio is important for all cells and especially critical for plant cells.
Because of limited water supplies and the widespread use of irrigation, the soils of many cultivated areas have become increasingly salinized. In particular, modern agricultural practices such as irrigation impart increasing salt concentrations when the available irrigation water evaporates and leaves previously dissolved salts behind. As a result, the development of salt tolerant cultivars of agronomically important crops has become important in many parts of the world; for example, in salty soil found in areas such as Southern California, Arizona, New Mexico and Texas.
Dissolved salts in the soil increase the osmotic pressure of the solution in the soil and tend to decrease the rate at which water from the soil will enter the roots. If the solution in the soil becomes too saturated with dissolved salts, the water may actually be withdrawn from the plant roots. Thus the plants slowly starve though the supply of water and dissolved nutrients may be more than ample.
Salt tolerant plants can facilitate use of marginal areas for crop production, or allow a wider range of sources of irrigation water. Traditional plant breeding methods have, thus far, not yielded substantial improvements in salt tolerance and growth of crop plants. In addition, such methods require long-term selection and testing before new cultivars can be identified.
Accordingly, there is a need to improve and/or increase salt tolerance in plants, particularly those plants that are advantageously useful as agricultural crops.